The present invention relates to DNA probes, a method and a kit for identifying antibiotic-resistant strains of bacteria.
The occurrence of antibiotic-resistant strains of bacteria, particularly of streptococcus strains, represents an increasing problem. So far, antibiotic susceptibility tests have been carried out by isolating bacteria and establishing a culture to define the minimum antibiotic inhibitory concentration in a biological test. This method takes at least 1 to 2 days. Well-calculated and thus optimum treatment is not possible within this period. Therefore, there is a need for a faster identification of existing resistances.
The object of the present invention consists in providing products and methods by means of which bacterial strains, particularly streptococcus strains, can be tested fast and reliably for existing antibiotic resistances.
This object is achieved by the subject matters defined in the claims.
The invention is described below by way of penicillin resistance of Streptococcus pneumoniae. However, this principle also applies in correspondingly general fashion to bacteria and resistances to other antibiotics. Neisserias and MRSA strains (methicillin-resistant Staphylococcus aureus), which do not produce xcex2-lactamase, are mentioned by way of example.
All of the penicillin-resistant S. pneumoniae strains have modified penicillin target proteins (penicillin-binding proteins, PBP). The DNA sequences of genes which play a decisive part in the development of penicillin resistance in Streptococcus pneumoniae have meanwhile been determined in a number of penicillin-resistant streptococcus strains. Three genes were identified where differences between sensitive and resistant strains occur in connection with the development of penicillin resistance: PBP2x, PBP1a and PBP2b.
A comparison between the DNA sequences shows within the genes regions which are present in all of the sensitive S. penumoniae strains but are modified in resistant strains. In this connection, reference is made to FIG. 1 which shows that the resistant strains differ more or less markedly from the sensitive strain R6 in the PBP2x gene but also differ among themselves.
Because of the above finding that differences between penicillin-sensitive and penicillin-resistant strains occur within certain genes, the applicant developed DNA probes by means of which resistant and sensitive strains can be differentiated. In this connection, reference is made to FIG. 4. The probes which are specific to sensitive sequences discriminate genes which code for low-affinity PBP variants responsible for penicillin resistance. The probes which are specific to resistant sequences react with a very frequently occurring class of PBP variants and can also be used for epidemiological purposes.
The applicant identified the following DNA probes [SEQ ID NOS.:1-8 ]:
a) Sensitivity-specific probes for PBP2x. The numerals in the column xe2x80x9cnucleotidexe2x80x9d refer to the nucleotides of the published sequence (Laible et al., Mol. Microbiol. 5, pp. 1993-2002 (1991)). The numerals in parentheses refer to the codon and the position (1, 2 or 3) within the codon of the structural gene. The number of bases in the nucleotide is given by xe2x80x9cmericxe2x80x9d.
b) Resistance-specific probes [SEQ ID NOS.: 14-17] for PB2x (as above; sequences in parentheses [SEQ ID NOS.: 20-23] are in accordance with the corresponding sections of sensitive strains).
c) Sensitivity-specific probes [SEQ ID NOS.: 9-12] for PBP1a (values refer to the nucleotides of the published sequence of the structural gene; Martin et al., EMBO J. 11, pp. 3831-3836 (1992))
d) Resistant specific probes [SEQ ID NOS.: 18-19] for PBP1a ([SEQ ID NOS.: 24-25] in parentheses).
e) Sensitivity-specific probes [SEQ ID NO.: 13] for PBP2b (values refer to the nucleotides of the published sequence of the structural gene; Hakenbeck, R., Matrin, C., Dowsen, C., Grebe, T., J. Bacteriol. 176, pp. 5574-5577 (1996))
N=any nucleotide
The above probes and those differing therefrom by one or several nucleotides, preferably up to 4 nucleotides, respectively, are perfectly suited to test unknown Streptococcus pneumoniae strains for resistance to penicillin.
For this purpose, bacteria according to the invention are centrifuged off a sample and in the case of S. pneumoniae the PBP genes (the resistance determinants) are amplified directly via PCR (polymerase chain reaction) as described in the literature (Grebe and Hakenbeck (1996), Antimicrob. Agents Chemother. 40, pp. 829-834). The advantage in connection with S. pneumoniae consists in that a detergence-induced lysis occurs rapidly and thus PCR can be carried out without long-winding DNA preparations. Since this step fails with other streptococci, only pneumococcus DNA is amplified specifically by means of this step. As an alternative, bacterial DNA (chromosomal and/or extrachromosomal) is isolated according to standard methods. This DNA is hybridized with at least one sensitivity-specific probe and with at least one resistance-specific probe under standard conditions with which a person skilled in the art is sufficiently familiar (see e.g. Maniatis et al., Molecular Cloning, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory). The hybridization is preferably carried out under stringent conditions such as 20xc2x0 C. below the melting point of the hybridizing DNA. The oligonucleotides are preferably chosen such that they have similar melting temperatures and thus several of them can be tested in the same hybridization batch under the same conditions (see FIG. 2). The oligonucleotides are preferably labeled when offered (P32, S35, biotin/avidin system; dioxygenine (DIG)-labeled; fluorescein-labeled) and hybridized against immobilized DNA. As an alternative, the oligonucleotides are offered on an oligonucleotide microarray in non-labeled fashion and the DNA to be hybridized is obtained via PCR and labeled while amplified.
It can be concluded from the hybridization result whether or not the unknown strain is sensitive to antibiotics. Depending on the resistance gene, at least one sensitivity-specific probe and one resistance-specific probe should be used for the hybridization. However, the DNA of the unknown strain is hybridized advantageously with several sensitivity-specific and resistance-specific probes in succession, since evaluation of resistance by means of only one combination of sensitivity-specific probes and resistance-specific probes can be inaccurate and rather only serve as a rough estimate. This applies particularly to the case of penicillin resistances in pneumococci and neisserias.
Preferred hybridization conditions depend on the AT content and length of the oligonucleotides. The person skilled in the art can select suitable conditions on the basis of his technical knowledge. Thus, e.g. 10-100 ng/ml labeled oligonucleotide for PBP2x (see above) are used in SSC hybridization solution at a hybridization temperature of 45xc2x0-60xc2x0 C. for at least 5 hours, preferably overnight.
The oligonucleotides can also be used as PCR primers to as to develop a PCR test therewith (see FIG. 3). This test can dispense with the somewhat more time-consuming hybridization. However, several PCRs must be used per strain. This method is suitable above all for epidemiological purposes.
The circumstance that less probes are known for PBP1a and particularly for PBP2b follows from the fact that smaller gene regions are of significance for resistance in PBP1a and particularly in PBP2b and therefore also only smaller regions have a sequence variation.
The invention also relates to a kit for carrying out the above method. This kit comprises means for isolating DNA from bacteria and for the PCR amplification of specific resistance determinants, respectively, sensitivity-specific DNA probes and resistance-specific DNA probes (lyophilized and as oligonucleotide microarray, respectively), reagents, solutions, buffers and means for hybridization and the subsequent detection of hybridized DNA. The sensitivity-specific DNA probes and resistance-specific DNA probes are preferably the ones listed above.
The advantage of the present invention is that bacteria, particularly pneumococci, can be assessed as to antibiotic resistance within the shortest time, i.e. within few hours. This enables subsequently a well-calculated and efficient treatment of diseased patients.